Overall objective of this research is to study the mechanism of membrane biogenesis an membrane selectivity. Interest in the study of sphingolipids has been stimulated by recent observations relating these lipids to intercellular recognition, immunological determinants, and ion transport. The specific purpose of this project will be to determine the mechanism of synthesis of sphingosine, and the in vivo pathway for the biosynthesis of sphingomyelin and lucosylceramide. In vivo studies will be done with hamster cells grown in tissue culture. We will determine the in vivo occurrence of postulated intermediates in the biosynthesis of sphingosine such as: 3 keto-spingosine and -dihydrosphingosine, 3 keto-sphingosine and -dihydrosphingosine containing-ceramides, -sphingomelins and of phytosphingosine. For those intermediates that are detected an attempt will be made to develop a cell free system for their synthesis. We will also determine whether the in vivo pathway for the biosynthesis of spingomyelin involves a condensation of CDP-choline with ceramides or sphingosylphosphoryl choline with a fatty acyl CoA derivate. We will examine whether the in vivo synthesis of glucosylceramide occurs via a condensation of UDP-glucose with ceramides or glucosylspingosine with a fatty acyl CoA derivate. These studies will constitute the groundwork for futher investigations on (1) subcellular localization of these enzymes, and (2) attempts to modify the sphingolipid composition of cells in culture, enabling one to correlate changes in composition with alterations in proposed functions ascribed to sphingolipids.